The Molecular Characteristics And Dissemination Mechanism Of NDM Producing Escherichia Coli In Shandong Province

[Background]Escherichia coli(E.Coli)is the most predominant pathogen causing community-acquired infections and nosocomial infections.Extended-spectrumβ-lactamases(ESBLs)and/or AmpC producing isolates spread rapidly all over the world.Carbapenems showed powerful activity against ESBLs and/or AmpC-producing isolates.Because of indiscriminate use of carbapenems,carbapenem resistant enterobacteriaceae(CRE)showed a trend of global dissemination.Because of the high mortality and rapid speed of spreading,CRE has become a global health crisis.Predictions of O’Neill J indicated that deaths caused by multidrug resistant E.coli expected to over 3 million each year by 2050.In a 2013 US CDC report,CRE were listed as one of the three most urgent antimicrobial resistant threats.Carbapenem resistance mainly arise from production of carbapenemase enzymes,changes in membrane permeability due to mutations in porins coupled with ESBLs and/or AmpC over expression.KPC、IMP、VIM、NDM are the most prevalent carbapenemases globally.KPC and IMP are the dominant carbapenemases in China.The genotype、phenotype.epidemiological studies and the mechanism research of CRE isolates are helpful to control outbreaks and spread of the multidrug-resistant organisms.We collected 23 single patient carbapenem resistant E.Coil(CREC)isolates in Shandong Provincial Hospital from January 2015 to December 2015.The study firstly revealed the mechanism and genotypes of carbapenemase E Coli isolates in Jinan.Shandong province.We explored the molecular epidemiological characteristics of CREC in the locality and provide guides for clinical treatments in the northern area of China.The study include five parts as follows.Part Ⅰ:Screening and in vitro antimicrobial susceptibility testing of CREC isolates;Part 2:Homology analysis of CREC isolates;Part 3:The molecular characteristics and dissemination mechanism of NDM producing Escherichia coli;Part 4:study on virulence factors of CREC isolates;Part 5:Rapid detection of NDM producers by MALDI-TOF MS.[Objective]1.To identify the CRE isolates by phenotypic screening tests and investigate the antimicrobial susceptibility of the strains.To analysis the homology of the CRE isolates.2.To reveal the molecular epidemiology and dissemination of NDM producing isolates.5.To study the correlation of virulence factors,genotypes and resistance genes.6.To establish the methodology of rapid detection of NDM producers by MALDI-TOF MS.[Materials and Methods]1.23 clinical isolated CREC strains and 20 carbapenem sensitive E.Coli(CSEC)strains were collected from Shandong Provincial Hospital.The isolates were re-identified using VITEK Compact system and MALDI-TOF MS.2.The isolates of antimicrobial resistance were detected by K-B test,and all antimicrobials breakpoints follow the CLSI(2016)interpretive criteria except tigecycline.The Interpretive criteria for tigecycline was according to EUCAST.Phenotypic confirmation test were performed,including Modified Hodge test,EDTA-disk synergy test,mCIM.Broth Microdilution method and ETEST was conducted to determine minimum inhibitory concentrations(MICs)of antimicrobials.3.The clonal homology was detected by MLST and PFGE.The results were analysed by softwares such as BioNumerics,eBURST and MEGA 6.4.Genotypes of ESBLs(including blaTEM,blaSHV,blaOXA,bsaCTX-M),Carbapenemases(including blaKPC,blaNMC,blasME,bla[M],blaGES,blaIMP,blaVIM,blaSPM,blaSIM,blaGIM,blaNDM,blaOXA),AmpC(blaACC,blaACT,blaBIL,blaCMY,blaDHA,blaFOx,blaLAT,blaMIR and blaMOX),Fosfomycin resistance(fosAfosC,FosA3),Quinolone resistance(qnrA.qnrB,qnrC,qnrD,qnrS,qepA,aac(6’)-Ib-cr),16S rRNA methylase(armA,npmA,rmtA,rmtB,rmtC and rmtD)and polymixin B resistance were determined by the PCR and sequence analysis of the amplification products.Outer membrane porins of the isolates absent of carbapenemases were analyzed by SDS-PAGE.5.Plasmid conjugation experiments were performed to determine mobility of plasmids haboring NDM genes.S1-PFGE of clinical isolates and conjugant strains were conducted to determine the sizes and amounts of plasmids.PBRT was conducted to analysis the types of plasmids carrying NDM genes.Stability of plasmid haboring NDM genes were tested by repeated streaking on agar without antibiotics for 10 times.6.All isolates were designated to one of E.coli phylogenetic groups(A,B1,B2,and D)through multiplex PCR-based method of Clermont et al.Virulence factors of 23 Escherichia coli isolates were compared among different types of extended-spectrum-lactamases(ESBLs)and different STs7.CHCA used as internal reference,we established a method validating the use of ertapenem molecules for the detection of carbapenemase activity by MALDI-TOF MS.The sensitivity and specificity were evaluated by testing 23 CREC isolates and 20 carbapenem sensitive E.Coli strains.[Results]1.A total of 1575 clinical E.Coli strains were isolated from January to December 2015 in Shandong Provincial Hospital,of which the carbapenem resistant E.Coli strains accounted for about 2.4%.In our samples,these CRE strains were mainly isolated from patients in urology ward 26.3%(10/38),pediatrics ward 15.8%(6/38)and ICU 15.8%(6/38).The main sources of carbapenemases producing E.coil were from urine47.2%(20/38).wounds11.3%(4/38),sputa 18.9%(7/38),abdominal specimen 15.79%(6/38).Among the 22 patients,7 patients used carbapenem or cephalosporin greater than 7 days and 21 patient were under invasive operations after admission.2.The antimicrobial susceptibility testing indicated that all the isolates were resistant to imipenem.meropenem,ertapenem.cephalosporin,piperacilin/tazobactam and quinolones.The resistance level of these antimicrobials were as high as MIC90 above 128μg/ml.All the isolates were sensitive to tigecycline and polymyxin B.Amikacin showed satisfactory activity also,with only 4 isolates resistant.The rate of fosfomycin-resistant strains were 30.43%(7/23).3.The result of PFGE and MLST showed clonal dissemination was present in our hospital.According to the Tenover’s standard,23 isolates belonged to 11 main clone clusters.Integration of foreign genes and genetic exchanges were present in the strains dissemination.MLST indicated that ST167(11/23),ST617(3/23),ST405(3/23),ST410(3/23)were the predominant STs of CREC.4.Detection of carbapenemases gene and DNA sequencing indicated that 20 isolates were NDM positive,with 6 isolates of NDM-1,12 isolates of NDM-5,1 isolate of NDM-7 and 1 isolate of NDM-3.Among the CRE strains,7 isolates carried AmpC genes and all the isolates carried ESBLs genes.CTX-M and TEM were the most common ESBLs genes.The CTX-M types were established by sequencing and BLAST.The primary CTX-M types were CTX-M-14(13/23),CTX-M-15(6/23),followed by CTX-M-55(6/23).Two or more ESBLs genes were detected in 16 isolates.Quinolone resistance genes were detected in 18 isolates,including 9 isolates carrying qnrB gene and 8 isolates carrying qnrS gene.7 isolates carrying qnrS gene were coupled with aac(6’)-Ib-cr gene.FosA3 attributed to the resistance for all the 7 fosfomycin-resistant strains.SDS-PAGE approved that the strains absent of carbapenemases gene lost at least one porin coupled with ESBLs and/or ApmCs.5.S1 enzyme digestion PFGE showed that at least one plasmid was contained in the clinical strains.15 strains contained the 50 kb plasmid which maybe the prevalent plasmid haboring carbapenemases gene.17 transconjugants were obtained after selection on agar with ertapenem and sodium azide,respectively.The antimicrobial susceptibility testing showed that transconjugants were resistant to imipenem,meropenem and ertapenem.S1 enzyme digestion PFGE of the transconjugants showed that 11 isolates carried 50 kb plasmid,7 isolates carried the 70-80 kb plasmid and 2 isolates carried the 120-130 kb plasmid.3 transconjugants carried 2 plasmids which meant the plasmids haboring carbapenemases gene were compatible and transferable.Although plasmid conjugation and transformation experiments were performed several times,3 CREC failed to obtain transconjugant.We presumed that may because the carbapenemase genes locate on chromosome or the plasmid bearing carbapenemase genes were not transferable.Typing of the plasmid was performed by multiplex PCR and 14 plasmids were determined as IncX3.Plasmids haboring carbapenemase genes were stable in isolates proved by plasmid stability experiments.Phylogenetic analyses showed that our strains fallen into three main phylogenetic groups(16 isolates were B1 group,3 isolates were D group,4 isolates were A group).All of the ST405 strains were D group and all of the ST410 were B1 group.7 isolates of ST167 were B1 group and 4 isolates were A group.B2 group exhibited several virulence factors was not detected in this study.10 strains carried capsule polysaccharide synthesis genes(cps).Pyelonephritis-associated pili(Pap G and papC)were detected in 20 isolates.including 19 isolates haboring papG 11,11 isolates haboring papG Ⅲ,and 3 isolates haboring papC.9 isolates carried the serotype fimbriae M gene and 3 isolates carried pathogenicity associated islands(PAI)gene.Compared with the CTX-M-55 producers,the CTX-M-15 producers were more likely to reserve iroN gene and less possibility to reserve papG II gene.TEM-1 producers were most possible to carry papG II genes in all the ESBLs strains.7.Ertapenem used as substrates,the study established a quick and accurate method to detect NDM-producing isoaltes by MALDI-TOF MS,with 100%specificity and 100%susceptibility.[Conclusions]1.These CREC strains were mainly isolated from urology.pediatrics and ICU ward.The predominat sources of the isolates were from urine.wounds.sputa and abdominal specimen.Risk factors for CREC infections were usage of carbapenem or cephalosporin greater than 7 days and invasive operations.It is necessary to strengthen the monitoring of nosocomial dissemination of CREC.2.The antimicrobial susceptibility testing indicated that all the isolates were resistant to multidrugs including β-lactam antibiotics and quinolones.But all the isolates were sensitive to tigecycline and Polymyxin B.Amikacin and fosfomycin showed high activity,also.3.Clonal dissemination was present in our hospital.The predominat strains were ST167,ST617,ST405 and ST410.Gene exchanges and integrations were observed through comparations of the PFGE finger-prints of strains.4.NDMs were the main carbapenemase detected in our strains.The predominat subtypes of NDM were NDM-1 and NDM-5.Loss of porin coupled with production of ESBL and/or AmpC was another mechanism of CRE.The primary CTX-M types were CTX-M-14 and CTX-M-15.QnrB and qnrS were the predominat quinolone resistant genes.16S rRNA methyla was detected in several strains which mediated resistance to amikacin.5.The most domain NDM-carrying plasmids were typed as IncX3 sized from 50 kb to 130 kb.The plasmids were compatible,transferable and stable in the isolates.6.Our E.coli strains were fallen into three main phylogenetic groups,including B1,A and D.Cps,papG,papC,serotype fimbriae M,iroN played a major role in the evolution of pathogenicity.7.MALDI-TOF MS showed perfect specificity and susceptibility in detection of NDM-producing isoaltes.